THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 33, Issue of November 25, pp. 25132”25138,1993 Printed in U. S. A.

1,25-Dihydroxyvitamin D3 Activates Raf Kinase and Raf Perinuclear Translocation via a Protein Kinase C-Dependent Pathway*

(Received for publication, May 22, 1993)

Trevor W. Lissoos$, David W. A. Beno, and Bernard H. Davis$ From the Gastroenterology Section, Department of Medicine, University of Chicago, Chicago, Illinois 60637

1,26-Dihydroxyvitamin D3’s (D3) potential mito- genic mechanism of action was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which pro- liferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3’s enhancement of [‘Hlthymidine incorporation was associated with nuclear Egr expression. ,Recent studies have causally linked the activated proto-oncogene c- Raf with downstream Egr induction. The serine-thre- onine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma mem- brane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfam- ily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the acti- vation of Raf was examined following D3 exposure. D3 induced Raf activation as assessed via (a) enhanced Raf phosphorylation following in vivo ’‘P labeling, (b ) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and (c) the shift in Raf physical localization changing from a diffuse cytoplasmic dis- tribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cas- cade leading to Raf kinase activation may involve a protein kinase C-dependent pathway, since vitamin D- stimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or stauro- sporine pretreatment.

1,25-Dihydroxyvitamin D3 (D3)’ has been identified as a major differentiation agent in a variety of cell types (1). Its biphasic effects with regards to cell proliferation (i.e. both stimulatory and inhibitory) as well as its variable effects on gene transcription are incompletely understood, although the

* This work was supported in part by the Liver Research Fund, University of Chicago and National Institutes of Health Grants DK40223, DK 42086, and DK 07074-18. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Current address: Gastroenterology Division, Dept. of Medicine, Washington University School of Medicine, 660 S. Euclid, P. 0. BOX 8124, St. Louis, MO 63110.

To whom correspondence should be addressed: Gastroenterology Section, Dept. of Medicine, MC 4076, 5841 S. Maryland Ave., Uni- versity of Chicago, Chicago, IL 60637. Tel.: 312-702-1467; Fax: 312-

The abbreviations used are: D3, vitamin Da; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PDGF, platelet-derived growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

702-2182.

nuclear vitamin D receptor is generally regarded as playing a central role (1). This latter receptor is a member of the steroid superfamily of receptors and the prenuclear events leading to receptor interactions have not been well explored (1). How- ever, recent studies involving intact isolated colonic epithelia as well as CACO-2 intestinal cell cultures suggest that D3 can cause acute changes in intracellular calcium and inositol 1,4,5- triphosphate formation (2, 3). The dominant mechanism(s) of action for these nongenomic effects are unknown, yet they may be distinct from the better studied genomic effects, since preliminary work has also found that vitamin D analogues with markedly different affinities for the nuclear vitamin D receptor have similar effects on intracellular calcium (4). As inositol 1,4,5-triphosphate formation is often associated with protein kinase C (PKC) metabolism, it is possible that PKC could play a key role in these nongenomic effects (5). Fur- thermore, as no plasma membrane receptor for D3 has been identified, it is possible that PKC directly or indirectly could play this role. Using the HL-60 promyelocytic leukemia cell line, previous studies have in fact shown that D3 can up- regulate the rate of transcription of PKC as well as the endogenous phosphorylation of PKC substrates without ex- ogenous activation or endogenous diacylglycerol formation (6). In addition, D3 modulation of HL-60 differentiation and c-myc transcription is blocked by inhibitors of PKC (7). These studies were performed over a 4-24-h time course, and there- fore, the acute effects of D3 and the mechanistic role of PKC in this pathway are unknown (6,7). Recent preliminary work, however, in CACO-2 cells has found that phorbol 12-myristate 13-acetate (PMA) induced down modulation of PKC-a en- hanced D3’s acute (in minutes) induction of cytosolic calcium, suggesting a role for PKC in the negative feedback regulation of D3-induced calcium rises (5).

These mechanistic studies have largely been done in malig- nant derived cell lines. However, D3’s physiologic role relates to its traditional in vivo kidney, intestine, and bone targets as well nuclear D3 receptor expression in heart, brain, and breast tissue (1). In addition, recent studies have found D3 mitogenic effects for circulating monocytes as well as freshly isolated aortic tissue and cultured vascular smooth muscle cells (8, 9). To extend the potential range of D3 targets as well as to explore prenuclear D3 pathways, the current study examined D3’s effects on rat hepatic sinusoidal Ito cells. This cell type undergoes activation and proliferation during liver injury and fibrogenesis in vivo and is a major source of the extracellular collagen matrix which accumulates during the cirrhotic process during the formation of constrictive nodules (10). The factors that mediate and perpetuate this pathologic activation are poorly understood, but they remain a central issue in ultimately understanding and regulating the fibro- genic process.

In the current work, D3 was found to be mitogenic for Ito cells and to induce nuclear Egr expression. To examine po-

25132

Vitamin D3 Activates Raf Kinase 25133

tential proximal mediators, the activation of the cytoplasmic proto-oncogene c-Raf, a serine threonine kinase, was exam- ined as it has been shown previously to activate Egr and has been identified as a key link between a variety of plasma membrane and cytoplasmic tyrosine kinases and nuclear tran- scriptional activators (i.e. fos, jun) (11, 12). D3 was found to activate Raf and Rafs associated kinase activity as well as induce Raf s physical perinuclear translocation. Similar acti- vation of Raf kinase function was found in 3T3 cells within 1 min of D3 exposure. Furthermore, the induction of Raf kinase was attenuated by pretreatment with PMA or the PKC inhib- itor, staurosporine, suggesting a central, likely upstream, role for PKC in D3-induced Raf activation.

MATERIALS AND METHODS

Chernicals-1,25-dihydroxyvitamin D3 (D3) (Steroids Ltd., Chi- cago) used in the experiments was stored as a stock solution in 100% ethanol under argon in a brown bottle a t 4 "C until use. For experi- ments, D3 was freshly diluted in tissue culture media. Control cultures treated with equivalent volumes of ethanol vehicle (final concentra- tion <0.1%) were indistinguishable from untreated cells. PDGF-BB was obtained from Amgen and Life Technologies, Inc. PMA and

use. [32P]Orthophosphoric acid (DuPont NEN) was mixed with phos- staurosporine (Sigma) were prepared freshly immediately prior to

phate-free (Life Technologies, Inc.) media just prior to use. Cell Culture-Hepatic Ito cells were isolated from Sprague-Dawley

male rats by previously described methods and subcultured on tissue culture flasks precoated with type I calf collagen (13, 14). 3T3 cells were maintained (courtesy of V. Sukhatme) on uncoated flasks. Experimental manipulations were performed with cells maintained on either 24-well or 75-cm2 precoated plates. Cellular quiescence was induced by 24 h culture in 0.4% fetal calf serum (FCS) (Ito cells) or 0.1% FCS (3T3 cells).

Cell Proliferation-Ito cells (groups of four parallel wells) were cultured on 24-well plates under subconfluent conditions in 0.4% FCS for 24 h. The medium was then replaced with fresh medium contain- ing D3 or PDGF (10 ng/ml). Eight hours later, the cultures were pulsed with [3H]thymidine and incubated for an additional 16 h. These conditions were previously found to be optimal for assessing the Ito cell mitogenic response (13-15). The degree of labeling was found to correlate with changes in cell number as well as in situ bromodeoxyuridine nuclear labeling (14, 15).

Raj Actiuation-The c-Raf serine/threonine kinase activation which occurs upon exposure to a variety of mitogens, including PDGF, is due to tyrosine, serine, and threonine phosphorylation (11). The cells were preincubated in phosphate-free media containing "P- labeled phosphoric acid (0.2 mCi/ml) for 3 h at 37 "C prior to D3 or PDGF stimulation. Phosphate-free media had no apparent effect on cell morphology or viability over a 24-h period of observation. It0 cell lysates k D3 (or PDGF) were processed in RIPA (50 mM Tris/HCl, pH 7.5, 150 mM NaCI, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 2 mM EDTA, 10 mM sodium fluoride, 10 pg/ml leupeptin, 10 pg/ml aprotonin, 300 pg/ml phenyl- methylsulfonyl fluoride) buffer and centrifuged at 12,000 X g X 15 min to remove insoluble cell material. The lysates were then immu- noprecipitated X 3 h a t 4 "C using monoclonal PBBl Raf antibody purified from mouse ascites with a protein G column (16). The characteristic 72-kDa Raf protein identified by the mono-specific antibody is completely blocked by preincubation with the Raf protein (16). Immunoprecipitation was performed with protein A-agarose beads prebound with a horse anti-mouse IgG (Vector) linker. The Raf antigen-antibody complexes were subsequently washed 2 X in RIPA, eluted with 4 X solubilization buffer (4% SDS, 10% glycerol, 50 mM Tris/HCl, pH 6.8), heated to 95 'C X 3 min, and then centrifuged at 6500 X g X 4 min to remove the beads. The samples were then resolved electrophoretically a 7% SDS-gel. The gel was fixed, stained, dried, and directly exposed to Kodak XAR-5 film and intensifying screens at -70 "C.

Raf kinase function was assessed using the Raf-bound protein A- agarose immunoprecipitates obtained after a 3-h incubation as de- scribed previously (11, 16). In brief, the complexes were washed 2 X in RIPA, 1 X in kinase buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCI, 10 mM MnC12, 0.2 mM dithiothreitol, 1% Triton X-100) and incubated X 2 min at 37 'C in kinase buffer with [32P]ATP (10 pCi/ reaction) and histone 1 protein (24 pg/reaction; Worthington) as an

exogenous substrate for phosphorylation. The reaction was termi- nated by adding 4 X solubilization buffer, heated to 95 "C X 3 min, and processed as described above. Eluates were loaded on a 14% SDS- gel and the gel containing the resolved proteins was subsequently fixed, stained, dried, and exposed to Kodak XAR-5 film. Relative kinase activity was determined by scanning the histone-labeled band with a laser densitometer. Representative kinase assays are shown and were repeated in two to three separate experiments. TO suppress protein kinase C activity, the cells were either pretreated with PMA (800 nm) X 20 h or the PKC inhibitor, staurosporine (20 nm) X 1 h. When the acute effects of PMA were compared, Ito cells were exposed to PMA (100 nm) X 15 min.

The Raf cytoplasm to perinuclear translocation was assessed dur- ing Ito cell culture in response to D3 as described previously for It0 cell serum or PDGF stimulation (17). This enabled an assessment of the capacity of the activated Raf protein to physically shift its localization which might be important in the further downstream transmission of the mitogenic cascade. Ito cells were cultured in 24- well plates and made quiescent with overnight culture in 0.4% FCS as described above and then exposed to D3 for 30 min. The cells were then washed with iced PBS X 2, fixed in methanol X 10 min, washed sequentially in PBS followed by PBS + 1% bovine serum albumin (PBS/BSA), and blocked in normal horse serum (1:lO dilution in PBS/BSA) for 10 min. All incubations were performed at room temperature. The wells were then incubated with PBBl (5 pg/well) diluted in PBS/BSA for 1 h. Control wells either excluded the primary antibody or used equal amounts of purified mouse IgG. Following three washes with PBS/BSA, the wells were incubated with a biotin- ylated horse anti-mouse secondary antibody (Vector) for 1 h. After three additional PBS/BSA washes, the wells were incubated with the avidin-biotin peroxidase complex (Vector) for 45 min, and the final brownblack reaction product was produced using diaminobenzidine as described previously (15). The cells were viewed under Hoffman optics on an inverted Olympus microscope and photographed with technical pan (Kodak) film.

Egr Expression-To determine whether the D3-initiated cascade ultimately produced nuclear events generally associated with the initiation of mitogenesis and the Go to GI shift, the enhanced expres- sion of the nuclear Fos and Egr-1 proteins was determined immuno- cytochemically following D3 exposure. Stimulation with 20% FCS was included as a control stimulation group. Immunocytochemical staining was performed using the same technique described above with minor modifications. Cell fixation was achieved with either methanol alone X 10 min (Fos staining) or methanol/acetone (50/50; v/v) X 10 min (Egr staining). Primary antibody staining utilized a rabbit polyclonal Fos antiserum (3 pg/well; Oncogene Science) X 1 h at room temperature or a rabbit polyclonal Egr-1 antiserum (R5232) (1:500, courtesy of V. Sukhatme, University of Chicago) X 1 h at room temperature (18). Control staining included comparable amounts of rabbit IgG.

Statistical Analysis-Differences between the means of various subgroups were assessed by Student's t test and one-way analysis of variance using the Statworks statistical package.

RESULTS AND DISCUSSION

Quiescent cultured Ito cells exposed to either lo-' M or 10"' M D3 demonstrated a 1.6-fold increase in [3H]thymidine incorporation (n = 3, p < 0.01), whereas there was no signif- icant effect at lo-* M or lo-" M (Fig. 1 A ) . This concentration range is consistent with numerous in vitro studies and ap- proaches the physiologic range of D3 (1). The inability to augment DNA synthesis at the pharmacologic lo-' M dose is consistent with other studies which found that high doses of D3 may be antimitogenic (1). The degree of D3-induced stimulation does not reach the range induced by serum or PDGF, suggesting that D3's role in. vivo might be more as a co-mitogen (14, 15). Recent preliminary studies have dem- onstrated the PDGFD receptor mRNA transcript dramatically increases i n vivo in Ito cells acutely following liver injury suggesting this potent mitogen could play a key role during the Ito cell's proliferative activation response (19). D3's po- tential co-mitogenic role with PDGF has been demonstrated previously with vascular smooth muscle cells (8). Ito cells exposed to both D3 and PDGF also demonstrated a near-

25134 Vitamin 0 3 Activates Raf Kinase

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0

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FIG. 1. Vitamin D3-enhanced Ito cell DNA synthesis. Quiescent Ito cells cultured in 0.4% fetal calf serum were exposed to vitamin D3 for 24 h, and [3H]thymidine was added during the final 16 h to measure DNA synthesis. In B, PDGF-BB (10 ng/ml) was added alone or simultaneously with vitamin D1 (lo-' M) (n = 3-4).

additive mitogenic response uersm either stimulant alone (Fig. 1B).

Although mitogenic stimulation generally correlates with immediate early and/or proto-oncogene expression, previous work examining D3's induction of myc and Fos have found variable results (7, 9). In different cell types, D3 has been shown to either increase or decrease myc mRNA and D3 enhancement of [3H]thymidine incorporation has been asso- ciated with a decrease in c-Fos mRNA levels (7, 9). Part of these differences may relate to the simultaneous differentiat- ing effects of D3 which may be distinct from its mitogenic effects, but it also underscores the difficulties of extrapolating between different cell types given the apparent complexities of the D3 response (7-9). Exposure of Ito cells to D3 (lo-' M ) induced the nuclear expression of the immediate early gene,

egr, as shown in Fig. 2, 90 min following stimulation. This pattern of Egr induction parallels that observed for serum or PDGF stimulation in Ito cells and numerous other cell types undergoing mitogenesis (20). Previous work has shown that this results from the transient induction of egr mRNA tran- scription and de nouo Egr protein synthesis (20). Interestingly, while Egr induction has generally paralleled fos induction, Ito cells exposed in parallel to D3 failed to show nuclear Fos expression. Control cells stimulated with fetal calf serum displayed both Egr and Fos nuclear expression. This differ- ential D3 effect on Egr uersus Fos expression is also in contrast to PDGF's effect on human and rat Ito cells where both proteins are induced (17, 21).

To begin to consider the complex signal transduction events which may precede D3's nuclear effects represented by Egr

Vitamin D3 Activates Raf Kinase 25135

&.;- . - ' --a:' .'

FIG. 2. Fos and Egr immunolocal- ization. Passaged Ito cells cultured in '8, 0.4% fetal calf serum were exposed to vitamin DJ (IO-' M) (B, E ) or 20% fetal . . 4 calf serum (C, F) for 90 min or were left untreated (A, D). Following fixation, the cells were stained with either a D O ~ V - crl) clonal ~ g r ( A , B, C) or F O ~ (E, F ) 'anti- s '" r" serum or normal rabbit serum (D). An- y.: :*:;,, , ;. tigen localization was assessed following staining with a biotinylated secondary antibody, avidin biotin peroxidase com- . . plex, and diaminobenzidine to yield a : . . . * # .. /& - brown/black reaction product. Final IF ' . +* magnification: X 270. *.: .'; . .a -.

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induction, the activation of the cytoplasmic proto-oncogene c-Raf was examined. This serine-threonine kinase was con- sidered because transfection experiments utilizing plasmids which produce constitutively activated Raf have demon- strated downstream Egr activation (22). Similar studies uti- lizing plasmids to generate dominant negative Raf mutants block this effect as well as serum-induced mitogenesis in some cell lines (11). Raf in general has been implicated in a central signal transduction role at the cytoplasmic level upstream of mitogen-activated protein kinase but downstream of receptor tyrosine kinases (i .e. PDGF-R, EGF-R, NGF-R, T cell recep- tor, insulin receptor) and other cytoplasmic secondary mes- sengers (i.e. Src, Ras, protein kinase C). Previous Ito cell studies have demonstrated Raf activation following either PDGF or serum stimulation (17). Following in vivo 32P label- ing and Raf immunoprecipitation (Fig. 3A), Ito cells exposed to lo-' M D3 displayed the characteristic shift in Rafs elec- trophoretic mobility associated with its activated state and enhanced phosphorylation. This paralleled the effect induced by PDGF-treated cells. Raf phosphorylation correlates highly with enhanced kinase activity (11). D3 exposure enhanced Raf kinase activity 5-30 min following stimulation as assessed by the capacity of immunoprecipitated Raf to phosphorylate the exogenous histone 1 substrate (Fig. 3B). Since Rafs downstream effects may depend upon its physical perinuclear translocation, D3's alteration in Raf distribution was assessed immunocytochemically. As shown in Fig. 4, 30 min following D3 exposure, Rafs distribution had shifted from a diffuse

i

cytoplasmic locale to a predominantly perinuclear domain. The same apparent increase in its relative intensity following translocation has been observed in serum and PDGF-stimu- lated Ito cells and is likely related to a focal concentrating effect (11).

D3 induction of Raf activation resembles the previously mentioned external stimuli known to activate Raf (11). How- ever, these other stimulants generally have defined receptor complexes with related tyrosine kinase pathways (11). While their direct versus indirect role in phosphorylating and hence activating Raf may vary and is still a subject of investigation, D3 is generally felt to rely upon distinctly different pathways associated with steroid-like mediators (1). However, by link- ing D3 exposure to Raf activation, the current work implies that these two differing transduction pathways may now share several central features. Future work will need to consider whether other steroid mediators share the capacity to use Raf-related transduction paths. In this regard, it will be especially useful to identify the more proximal molecules which mediate D3 activation of Raf.

To begin this type of analysis, Ito cells were treated with PMA (800 nM X 20 h). Pretreatment with PMA has been shown recently to suppress Ito cell PKC protein production as in numerous other cell types (23). When D3 M) was then applied for 30 min and Raf kinase function assessed, PMA pretreatment was found to abolish the induction of activity observed in the cells exposed to D3 in the absence of PMA (Fig. 5 ) . PMA treatment X 24 h caused no apparent

25136

A. C

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68 -

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43 -

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Vitamin D3 Activates Raf Kinase

PDGF Vit D -

5' 15' 30' Vitamin D

FIG. 3. Vitamin Ds-induced Ito cell Raf activation. Quiescent Ito cells maintained in 0.4% serum were exposed to vitamin D3 (lo-' M) for 5-30 min as indicated or PDGF-BB for 15 min. In A , the cells were precultured in phosphate-free media for 3 h containing ["PI phosphoric acid (0.2 mCi/ml). The cells were then washed, scraped into RIPA buffer, and centrifuged to remove insoluble cell material. After protein normalization, aliquots were subjected to 3-h immunoprecipitation with monoclonal PBBl Raf antisera bound to horse anti-mouse- linked protein A-agarose. In A , the proteins were resolved on a 7% SDS-polyacrylamide gel, fixed, stained, dried, and exposed to photographic film. The Arrow on the left indicates phosphorylated Raf band following stimulation. In B, the Raf kinase assay was performed with the immunoprecipitates (X 2 min a t 37 "C in kinase buffer with histone H1 and [32P]ATP). The samples were eluted with solubilization buffer a t 95 "C X 3 min and resolved on a 14% SDS-polyacrylamide gel. The phosphorylated histone band (arrow) reflects kinase activity as is summarized graphically on the right uersus D3 exposure time (mean 2 S.D., n = 2).

A B

FIG. 4. Vitamin Ds-induced Raf translocation. The Raf pro- tein was localized in quiescent Ito cells ( A ) and 30 minutes following vitamin Da exposure ( B ) using the PBBl Raf monoclonal antibody (see text). Note shift from diffuse distribution in A (straight arrows) to a perinuclear localization in B (curved arrows). Final magnifica- tion: X 130.

loss of cell viability or any change in cellular morphology. Acute PMA treatment (100 nm) x 15 min activated Raf kinase as described in other cell systems (11,26). In parallel, Ito cell pretreatment with the PKC inhibitor, staurosporine, abol-

ished the vitamin D-induced activation of Raf kinase. To extend and generalize these findings, 3T3 cells were exposed to D3 over 1-10 min (Fig. 6). Raf kinase activity was markedly enhanced following 1 min of exposure, and this was sustained for 10 min. At 15- and 30-min time points, the activity returned to base line (data not shown). Parallel cultures pretreated with staurosporine showed attenuated Raf kinase activity. PMA treatment of Ito cells has been shown to reduce the PKC a, 6, and 6 isoforms, but it is probable that the observed suppressive effect is due predominantly to the re- duction in the a isoform (23, 24). Recent work utilizing recombinant baculoviruses expressing various PKC and R a f polypeptides in insect cells identified the conventional PKC types (including the (Y isotype) but not the novel types (in- cluding the 6 isotype) as capable of activating Raf in a tumor promoter 12-0-tetradecanoyl phorbol 13-acetate-dependent way (24). The current finding of D3-induced Raf stimulation and PKC dependence is similar to previous work involving the T cell receptor and Raf activation (25). Previous studies utilizing 3T3 cells and PDGF stimulation suggested that there are PKC-independent pathways leading to Raf activation as well (26). The exact mechanism of PDGF and D3's additive

FIG. 5. Ito cell Raf kinase suppression by phorbol ester and staurosporine. Ito cell immunoprecip- itates were obtained from quiescent cells (control) uersm cells treated with D3 X 30 min (vitamin D ) , cells treated X 20 h with PMA (800 ng/ml) and then exposed to D3 (PMAIVit D ) , cells treated with staurosporine (20 nM) for 1 h and then vitamin D (stauroloitarnin D ) , cells treated with PMA (800 ng/ml) X 20 h and then exposed to PMA (100 nm) for 15 min ( P M A I P M A ) , or cells exposed to PMA (100 nm) for 15 min ( P M A ) . Raf kinase assay was performed with histone H1 as described in Fig. 3 ( n = 2-3). Inset displays representative histone bands used for quantitation.

Vitamin D3 Activates Raf Kinase 25137

P

1.5 1

P

Staurosporlne

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FIG. 6. 3T3 cell D3-induced Raf kinase activity. 3T3 cell Raf immu- noprecipitates were obtained from quies- cent cells (control) uersus cells treated with vitamin Ds for 1 min (uit D X I ’ ) , 5 min (uit D x 5 ’ ) , 10 min (u i t D xIO’), or cells pretreated with staurosporine (20 nM) X 1 h followed by vitamin Ds for 1 min (stauroluit D X ] ’ ) or 5 min (staurol uit Dx5’). Raf kinase assay was per- formed with histone H1 as described in the legend to Fig. 5, and representative histone bands used for quantitation are shown as an inset ( n = 2-3).

25138 Vitamin D3 Actiuates Raf Kinase

Ito cell mitogenic effect described herein remains to be deter- mined, but it may relate in part to the potential use of both PKC-dependent and PKC-independent mechanisms of Raf activation. Nevertheless, the dependence on PKC function agrees well with the previously mentioned studies utilizing intestinal-derived cells which demonstrated D3-induced PKC translocation (2). It remains unclear how D3 enters the cell and interacts with the PKC molecule, but it is possible that PKC represents a major proximal signal receptor in mediating vitamin D-induced cellular proliferation as well as cellular differentiat~on as suggested by previous studies in HL60 cells (7).

Cumulatively, these studies provide further evidence that vitamin D, in addition to acting via intranuclear receptor binding and regulation of gene transcription, can involve prenuclear nongenomic pathways. Future studies are needed to better define the precise role of Raf (and mitogen-activated protein kinase) in mediating the distal nuclear events gener- ated by vitamin D, but the current work now emphasizes that the Raf/MAP pathway should be considered when examining the vitamin's mechanism of action.

A c k ~ ~ ~ d g ~ e ~ t s - W e thank Dr. T. Brasitus for supplying vita- min D as well as advice and en~ouragement during this study. We thank Dr. U. Rapp (National Cancer Institute) and Dr. V. Sukhatme (University of Chicago) for supply of antisera.

REFERENCES 1. Reichel, H., Koeffler, H. P., and Norman, A. W. (1989) N. E&. J. Med.

320,980-991

2. Wali, R. K., Baum, C. L., Sitrin, M. D., and Brasitus, T. A. (1990) J. Clin.

3. Tien, X. Y., Katnik, C., Nelson, D. J., Sitrin, M. D., and Brasitus, T. A.

4. Tien, X. Y., Brasitus, T. A., Norman, A. W., and Sitrin, M. D. (1992) Clin.

5. Tien, X. Y., Bissonette, M., Niedziela, S., Ro , H., Sitrin, M. D., and

6. Obeid L. M., Okazaki, T., Karolak, L. A., and Hannun, Y. A. (1990) J.

7. Sim son, R. U., Hsu, T., Wendt, M. D., and Taylor, J. M. (1989) J. Biol.

8. Mitsuhashi, T., Morris, R. C., and Ives, H. E. (1991) J. Ctin. Invest. 87,

Inuest. 8 6 , 1296-1303

(1992) Gastroenterology 102 , A581

Res. 40,265A

Brasitus, T. A. (1993) Gastroenterology 104, x649

Biot Chem. 265,2370-2374

CLm. 264,19710-19715

119-114

9.

10. 11. 12.

13. 14.

Drittanti, L., De Boland, A. R., and Boland, R. (1989) Bioehim Bwphy.

Laskin, D. L. (1990) Semis. Liuer DL 10,293-304 Rapp, U. (1991) Onco ene 6,495-500 Smeal, T., Binetru fs., Mercola, D., Grover-Bardwick, A., Heidecker, G.,

Davis,%. H. (1988) J. Cell Physml. 136,547-553 Davis, B. H., Kramer, R. T., and Davidson, N. 0. (1990) J. Clin. Inuest.

.AI ""

Acta 1014,112-119

f+p , U R., anzKarin, M. (1992) Mol. Cell. Bhl. 12,3507-3513

Re. 2M2-707n 15. Davis, B. H., Rapp, U. R., and Davidson, N. 0 . (1991) Biochern. J. 2 7 8 ,

- - , - - - - - - . - 43-47

16. Koiih- W., Weissin er E. Mischak, H. Troppmair, J., Showalter S. D., Llo;d, P., Heidecfer: G.: and Rapp, U: R. (1990) Oncogene 6, 713'-720

17. Beno, D. W. A., and Davis, B. H. (1992) Hepatology 16 , 157A 18. Cao, X. Koski, R. A,, Gashler, A., McKiernan, M., Morris, C. F., Gaffney,

R., day, R. V., and Sukhatme, V. P. (1990) Mol. Cell. Biol. 10, 1931- 1939

19. Fri'e;i;lan, S. L., Yamasaki, G., Wong, L., and Buck, J. (1992) Hepatology 1 fi. 142A

20. Sukgatrne, V. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89.1-4 21. Davis, B. H., Colt, D., and Beno, D. W. A, (1992) Hepatolo y l 6 , 9 6 A 22. Qureshi, S. A., Rim, M., Bruder, J., Kolch, W., Rap , U , Akhatme, V. P.,

23. Kobayashi, Y., Britton R. S., O'Neil, R., Li, S. C. Y., and Bacon, B. R. 24. Sozeri, O., dllmer , K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., and

25. Sierrel. J. N.. Klausner. fl. D.. Ram. U. R.. and Samelson. L. E. (1990) J.

, ""

and Foster, D. A. (1991) J. B~ol. Chem. 266,20584-20597

(1992) He atology 16,143A

Stabel, S. (1992) Onco ene 7,2259-2262

Bioi Chem. 265,18472-11880- .

Natl. Acad. Sci. UPS. A. 85,8855-8859 26. Morrison, D. K., Ka lan, D. R., Rapp, U., and Roberts, T. M. (1988) Proc.